Method for the treatment of bladder cancer

ABSTRACT

Methods of treating bladder cancer using terconazole are disclosed herein. Terconazole can be administered as part of a comprehensive treatment program, which can also include chemotherapy, immunotherapy, radiation therapy and/or surgical treatment.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of Indian Application 4641/MUM/2015,filed Dec. 9, 2015, the contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is directed to a method of treating bladder cancerusing terconazole or a pharmaceutically acceptable salt thereof.

BACKGROUND

Bladder cancer is a life-threatening and progressive disease, whichusually begins in the lining of the epithelial lining (i.e., theurothelium) of the urinary bladder. Invasive bladder cancer may spreadto lymph nodes, other organs in the pelvis (causing problems with kidneyand bowel function), or other organs in the body, such as the liver andlungs. Standard treatments for bladder cancer are surgery, radiationtherapy, chemotherapy, and biological therapy

Bladder cancer is diagnosed using cystoscopy and/or cytology, however,the latter is not very sensitive—a negative result cannot reliablyexclude bladder cancer.

There are newer non-invasive urine bound markers available as aids inthe diagnosis of bladder cancer, including human complement factorH-related protein, high-molecular-weight carcinoembryonic antigen, andnuclear matrix protein 22 (NMP22). NMP22 is also available as aprescription home test. Other non-invasive urine based tests include theCertNDx Bladder Cancer Assay, which combines FGFR3 mutation detectionwith protein and DNA methylation markers to detect cancers across stageand grade, UroVysion, and Cxbladder. The diagnosis of bladder cancer canalso be done with a Hexvix/Cysview guided fluorescence cystoscopy (bluelight cystoscopy, Photodynamic diagnosis), as an adjunct to conventionalwhite-light cystoscopy. This procedure improves the detection of bladdercancer and reduces the rate of early tumor recurrence, compared withwhite light cystoscopy alone. Cysview cystoscopy detects more cancer andreduces recurrence. Cysview is marketed in Europe under the brand nameHexvix.

The treatment of bladder cancer depends on how far cancer has spread.Most bladder cancer is found early, before it has spread into thebladder wall. Surgically, bladder cancer can be treated usingtransurethral resection of bladder tumor (TURBT), wherein the bladder isaccessed using a cystoscope passed through the urethra to removecancerous cells. In more severe cases, a partial or radical cystectomymay be performed. However, because of concerns regarding recurrence,patients often receive chemotherapy or immunotherapy in addition tosurgery. Chemotherapies which have been employed include methotrexate,vinblastine, doxorubicin, and cisplatin (MVAC), gemcitabine andcisplatin (GC). Administration of these drugs is often accompanied bysevere negative side effects.

Immunotherapies include intravesicular delivery of BacillusCalmette-Guérin (BCG). BCG is a vaccine against tuberculosis that isprepared from attenuated (weakened) live bovine tuberculosis bacillus,Mycobacterium bovis that has lost its virulence in humans. BCGimmunotherapy is effective in up to 66% of the cases at this stage, andin randomized trials has been shown to be superior to standardchemotherapy. The mechanism by which BCG prevents recurrence is unknown,but the presence of bacteria in the bladder may trigger a localizedimmune reaction which clears residual cancer cells. However, bladdercancer recurring in patients subsequent to BCG treatment is moredifficult to treat.

There remains a need for effective, non-surgical treatments of bladdercancer, including bladder cancer recurring post-BCG treatment. Thereremains a need for agents effective to treat bladder cancer with reducedside effect profiles relative to currently used medications.

SUMMARY

Disclosed herein are methods of treating bladder cancer, includingbladder cancer recurring post-BCG treatment. The methods includeadministering to a patient in need thereof terconazole in an amounteffective to treat the bladder cancer. In some instances, terconazolecan be administered as part of a combination therapy. Also disclosedherein are pharmaceutical compositions containing terconazole suitablefor the treatment of bladder cancer. In some instances, the compositionsinclude an additional anti-cancer agent.

The details of one or more embodiments are set forth in the descriptionsbelow. Other features, objects, and advantages will be apparent from thedescription and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 includes a depiction of a 2D assay of terconazole against humanbladder cancer cells.

FIG. 2 includes a depiction of a 2D assay of cisplatin against humanbladder cancer cells.

FIG. 3 includes a depiction of a 3D assay of terconazole against humanbladder cancer cells.

FIG. 4 includes a depiction of a 3D assay cisplatin against humanbladder cancer cells.

FIG. 5 includes a depiction of the anti-tumor efficacy of terconazole(CIPDE033) in combination with cisplatin in BXF 1036L; A) Modeled T/C,which is the mean of experimental T/C for each pair of conditions in thecombination matrix. B) Bliss index, which is the difference of Blissneutral and modeled T/C for each pair of conditions

FIG. 6 includes a depiction of the anti-tumor efficacy of terconazole(CIPDE033) in combination with cisplatin in BXF 1218L. A) Modeled T/C,which is the mean of experimental T/C for each pair of conditions in thecombination matrix. B) Bliss index, which is the difference of Blissneutral and modeled T/C for each pair of conditions.

FIG. 7 includes a depiction of the anti-tumor efficacy of terconazole(CIPDE033) in combination with cisplatin in BXF T-24. A) Modeled T/C,which is the mean of experimental T/C for each pair of conditions in thecombination matrix. B) Bliss index, which is the difference of Blissneutral and modeled T/C for each pair of conditions

DETAILED DESCRIPTION

Before the present methods and systems are disclosed and described, itis to be understood that the methods and systems are not limited tospecific synthetic methods, specific components, or to particularcompositions. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Ranges may be expressed herein as from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes

from the one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal embodiment. “Such as” is not used ina restrictive sense, but for explanatory purposes.

Disclosed are components that can be used to perform the disclosedmethods and systems. These and other components are disclosed herein,and it is understood that when combinations, subsets, interactions,groups, etc. of these components are disclosed that while specificreference of each various individual and collective combinations andpermutation of these may not be explicitly disclosed, each isspecifically contemplated and described herein, for all methods andsystems. This applies to all aspects of this application including, butnot limited to, steps in disclosed methods. Thus, if there are a varietyof additional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

In normal adult tissues, neovascularization is required only for repairof tissue injury. In malignant tissues, neovascularization is requiredfor tumors to grow beyond a certain size and to metastasize to newsites. A fine balance between stimulatory and inhibitory factorsproduced by the tumor and the surrounding stroma regulates new bloodvessel development. Bladder tumors produce high levels of multipleangiogenic stimulatory factors, including vascular endothelial growthfactor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8.Levels of these factors correlate with stage and outcome. Micro-vesseldensity, a surrogate marker for angiogenic activity, is a predictor ofdisease progression, vascular invasion, lymph node involvement, tumorrecurrence, and poor survival in cancer. Levels of VEGF and bFGF areinversely associated with prognosis. Based on these findings, it ishypothesized that targeting angiogenesis pathways alone or incombination with standard chemotherapeutic regimens in bladder cancerwill lead to improvement in patient outcomes.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that catalyzesoxidative degradation of cellular heme, liberating free iron, carbonmonoxide (CO) and biliverdin in mammalian cells. In addition to itsprimary role in heme catabolism, HO-1 exhibits anti-oxidative andanti-inflammatory functions via the actions of biliverdin and CO,respectively. HO-1 is strongly associated with various disease states,including cancer. Several lines of evidence have supported the role ofHO-1 in carcinogenesis and tumor progression. HO-1 deficiency in normalcells enhances DNA damage and carcinogenesis. Nevertheless, HO-1overexpression in cancer cells promotes proliferation and survival.Moreover, HO-1 induces angiogenesis through modulating expression ofangiogenic factors. Although HO-1 is an endoplasmic reticulum residentprotein, HO-1 nuclear localization is evident in tumor cells of cancertissues. It has been shown that HO-1 is susceptible to proteolyticcleavage and translocates to nucleus to facilitate tumor growth andinvasion independent of its enzymatic activity. HO-1 also impacts cancerprogression through modulating tumor microenvironments. Elevated hemeoxygenase-1 (HO-1) is associated with resistance to chemo- andradiotherapy through anti-apoptotic function. It has been reported thatHO-1 is significantly expressed in late stage bladder cancer tissuespecimen. High T stage (T2-4) correlated with higher percentage of HO-1positivity compared with low T stage (P=0.008) and high in patients withlymph node metastasis (P=0.022) than those without metastasis.Significant up-regulation of HO-1 expression is seen in moderate andheavy smokers compared to light or never smokers. Thus HO-1 can beconsider as ideal target site for effective treatment of bladder cancer.

Cancer cells exhibit elevated oxidative stress due to their highmetabolic rate. Moreover, they are surrounded by a complexmicroenvironment and significantly influenced by their interplays withthe stromal components, especially the infiltrating inflammatory cells.It is envisioned that the oxidative stress and stimulations by variousgrowth factors and cytokines released from stromal cells are capable ofinducing HO-1 gene transcription in tumor cells through activation ofvarious signaling pathways and transcriptional factors, including Nrf2,NF-κB, AP2 and others. Hypoxia has also been shown to induce HO-1expression. Furthermore, HO-1 gene expression is upregulated byoncogenes, such as Kaposi sarcoma-herpes virus and BCR/ABL kinase. Inaddition to the regulation at transcriptional level, HO-1 expression issubjected to post-transcriptional regulation. Regulation of HO-1 bymir378 is implicated in lung cancer growth and metastasis.Down-regulation of HO-1 by mir200c enhances the sensitivity of renalcancer cells to chemotoxic agents. Moreover, HO-protein is turnover byubiquitin-proteasome system. HO-1 is a physiological substrate of TRC8,which is an ER-resident E3 ligase associated with hereditary renal cellcancer and thyroid cancer. The tumor suppressive effect of TRC8 ismediated at least in part via targeting HO-1 for ubiquitination anddegradation in cancer cells.

Tumorigenesis is a multistep process in which the accumulation ofseveral genomic mutations is required to initiate the transformation ofnormal cells to become cancer cells. DNA damage caused by the reactiveoxygen species (ROS) is a major source of mutation. HO-1 down-regulationleads to the increase of ROS and DNA damage in cells. Furthermore, COimproves cell survival post irradiation or genotoxin treatment byinducing DNA repair. Therefore, increase in HO-1 expression prevents DNAdamage and the initiation of carcinogenesis in normal cells. However, atlate phase of tumorigenesis, HO-1 overexpression promotes cancer cellproliferation and invasiveness. HO-1 protects cancer cells fromapoptosis induced by chemotoxic agents or irradiation, suggesting itsinvolvement in therapeutic resistance. CO contributes to the resistanceof cancer cells to oxidative stress and chemotoxic agents by inhibitingthe heme-containing cystathionine β-synthase, which causes reducedPFKFB3 methylation and shift of glucose metabolism to pentose phosphatepathway, resulting in subsequent increase of NADPH to replenish reducedglutathione.

The immune/inflammatory cells recruited to tumor microenviroment haveprofound effects on cancer progression by modulating inflammatoryresponse and anti-tumor immunity. HO-1 modulates the immune regulatoryfunctions of myeloid cells by suppressing the expression ofpro-inflammatory cytokines, such as tumor necrosis factor-α, butpromoting the expression of immunosuppressive cytokine, interleukin-10(IL-10). HO-1 promotes inflammation-associated angiogenesis throughup-regulating VEGF expression in macrophages. Furthermore, HO-1expression in myeloid-derived suppressor cells participates in thesuppression of alloreactive T cells. Although HO-1 expression in thestromal macrophages has been seen in the cancer tissues, the impact ofHO-1 expression in myeloid cells on cancer progression is less explored.HO-1 expression mediates the immune suppressive function of a stromalmacrophage subpopulation expressing fibroblast activation protein-α.

Terconazole is a broad-spectrum antifungal drug effective as first linetherapy against candida species infections and used for the localtreatment of vulvovaginal candidiasis. Terconazole acts by binding toiron component of fungal cytochrome P450 enzyme lanosterol 14alpha-demethylase. Terconazole may be represented by the followingchemical formula:

Terconazole is sold as the racemic mixture of the (2S,4R) isomer(depicted above) and the enantiomeric (2R,4S) isomer.

It has been reported by in literature that ketoconazole and terconazoleinhibited rat spleen HO-1 activity with IC₅₀ of 0.3±0.1 μM and 0.41±0.01μM respectively.

Disclosed herein are methods of treating bladder cancer in a patient inneed thereof by administering an effective amount of terconazole. Unlessstated to the contrary, the term terconazole refers both to terconazolefree base and pharmaceutically acceptable salts thereof.

Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart undesirabletoxicological effects. Examples of such salts are acid addition saltsformed with inorganic acids, for example, hydrochloric, hydrobromic,sulfuric, phosphoric, and nitric acids and the like; salts formed withorganic acids such as acetic, oxalic, tartaric, succinic, maleic,fumaric, gluconic, citric, malic, methanesulfonic, ptoluenesulfonic,napthalenesulfonic, and polygalacturonic acids, and the like; saltsformed from elemental anions such as chloride, bromide, and iodide;salts formed from metal hydroxides, for example, sodium hydroxide,potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesiumhydroxide; salts formed from metal carbonates, for example, sodiumcarbonate, potassium carbonate, calcium carbonate, and magnesiumcarbonate; salts formed from metal bicarbonates, for example, sodiumbicarbonate and potassium bicarbonate; salts formed from metal sulfates,for example, sodium sulfate and potassium sulfate; and salts formed frommetal nitrates, for example, sodium nitrate and potassium nitrate.Pharmaceutically acceptable and non-pharmaceutically acceptable saltsmay be prepared using procedures well known in the art, for example, byreacting a sufficiently basic compound such as an amine with a suitableacid comprising a physiologically acceptable anion. Alkali metal (forexample, sodium, potassium, or lithium) or alkaline earth metal (forexample, calcium) salts of carboxylic acids can also be made.Pharmaceutically acceptable anions include the conjugate bases of theacids listed above.

Preferably, terconazole may be administered to the subject once daily,twice daily or thrice daily. A typical recommended daily dosage regimencan range from about 5 mg to 2,000 mg, from about 10 mg to 1,000 mg,from about 10 mg to 500 mg, from about 10 mg to 400 mg, from about 10 to200 mg, from about 10 to 100 mg, from about 10 to 50 mg, from about 50to 400 mg, from about 100 to 400 mg, or from about 200 to 400 mg.

Preferably, the active agent may be provided in the form of apharmaceutical composition such as but not limited to, unit dosage formsincluding tablets, capsules (filled with powders, pellets, beads,mini-tablets, pills, micro-pellets, small tablet units, multiple unitpellet systems (MUPS), disintegrating tablets, dispersible tablets,granules, and microspheres, multiparticulates), sachets (filled withpowders, pellets, beads, mini-tablets, pills, micro-pellets, smalltablet units, MUPS, disintegrating tablets, dispersible tablets,granules, and microspheres, multiparticulates), powders forreconstitution and sprinkles, transdermal patches, however, other dosageforms such as controlled release formulations, lyophilized formulations,modified release formulations, delayed release formulations, extendedrelease formulations, pulsatile release formulations, dual releaseformulations and the like. Liquid and semisolid dosage forms (liquids,suspensions, solutions, dispersions, ointments, creams, emulsions,microemulsions, sprays, patches, spot-on), parenteral, topical,inhalation, buccal, nasal etc. may also be envisaged under the ambit ofthe invention. The inventors of the present invention have also foundthat the solubility properties of the active agent may be improved bynanosizing thus leading to better bioavailability and dose reduction ofthe drug.

In one embodiment, terconazole may be present in the form ofnanoparticles which have an average particle size of less than 2,000 nm,less than 1,500 nm, less than 1,000 nm, less than 750 nm, less than 500nm, or less than 250 nm.

Suitable excipients may be used for formulating the dosage formaccording to the present invention such as, but not limited to, surfacestabilizers or surfactants, viscosity modifying agents, polymersincluding extended release polymers, stabilizers, disintegrants or superdisintegrants, diluents, plasticizers, binders, glidants, lubricants,sweeteners, flavoring agents, anti-caking agents, opacifiers,anti-microbial agents, antifoaming agents, emulsifiers, bufferingagents, coloring agents, carriers, fillers, anti-adherents, solvents,taste-masking agents, preservatives, antioxidants, texture enhancers,channeling agents, coating agents or combinations thereof.

Depending on the pathological stage, patient's age and otherphysiological parameters, and the extent of cancer progression,terconazole may require specific dosage amounts and specific frequencyof administrations. Preferably, the active agent may be administered atleast once, twice or thrice a day in an amount from 10 mg to 2,000 mg.In some embodiments, the active agent may be administered such that thetotal daily dose is in an amount from 10-1,000 mg, 50-1,000 mg, 50-750mg, 50-500 mg, 100-500 mg, 250-2,000 mg, 500-2,000 mg, 500-1,000 mg,250-1,000 mg, 250-500 mg, 1,000-2,000 mg, or 1,500-2,000.

Terconazole can be used to treat bladder cancers. In some embodiments,terconazole can reduce tumor size, inhibit tumor growth, alleviatesymptoms, delay progression, prolong survival, including, but notlimited to disease free survival, prevent or delay bladder cancermetastasis, reduce or eliminate preexisting bladder cancer metastasis,and/or prevent recurrence of bladder cancer.

As used herein, the term “delay” refers to methods that reduce theprobability of disease development/extent in a given time frame, whencompared to otherwise similar methods that do not include the use ofterconazole. Probabilities can be established using clinical trials, butcan also be determined using in vitro assays when correlations have beenestablished. In some embodiments, terconazole can inhibit bladder cancercell proliferation. For instance, at least about 10%, 20%, 30%, 40%,60%, 70%, 80%, 90%, or 100% of cell proliferation is inhibited. In someembodiments, terconazole can inhibit bladder cancer metastasis. Forinstance, at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%of metastasis is inhibited.

It can be preferable to diagnose the patient with bladder cancer priorto commencing the therapeutic methods disclosed herein. When thefunctional consequences are determined using intact cells or animals,one can also measure a variety of effects such as, in the case ofbladder cancer associated with tumors, tumor growth, tumor metastasis,neovascularization, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as cGMP. In the assaysof the invention, mammalian bladder cancer polypeptide is typicallyused, e.g., mouse, preferably human. Tumor cells release an increasedamount of certain factors (hereinafter “tumor specific markers”) thantheir normal counterparts. For example, plasminogen activator (PA) isreleased from human glioma at a higher level than from normal braincells. See, e.g., “Angiogenesis, tumor vascularization, and potentialinterference with tumor growth” pp. 178-184 in Mihich (ed. 1985)Biological Responses in Cancer Plenum. Similarly, tumor angiogenesisfactor (TAF) is released at a higher level in tumor cells than theirnormal counterparts. See, e.g., Folkman (1992) Sem Cancer Biol. 3:89-96.Different urine tests are available to look for specific substancesreleased by bladder cancer cells. One or more of these tests may be usedalong with urine cytology to help determine the bladder cancer. Theseinclude the tests for NMP22 (BladderChek) and BTA (BTA stat), theImmunocyt test, and the UroVysion test. In other instances, the patientcan be diagnosed with bladder cancer using cystoscopy. In certainembodiments, a patient having detectable amount of one or more of theabove markers, after receiving terconazole, with exhibit a 10%, 20%,30%, 40%, 60%, 70%, 80%, 90%, or 100% reduction in that marker.

Bladder cancer can be characterized by overall stage, 0-IV. Stage 0 isrefined by the letters a (designating non-invasive papillary carcinoma)and is (designating non-invasive flat carcinoma, which can be referredto as CIS). The stages can be further refined by one of threecategories: T categories refer to the extent the tumor has grown into orbeyond the wall of the bladder. T1 refers to cancer that has not growninto the muscle layers of the bladder. T2a indicates the cancer hasgrown into the inner half of the muscle layer, while T2b indicates theouter half of the muscle layer has been compromised. T3 indicates thetumor has grown into the fatty tissue surrounding the bladder (T3arefers to tumors that are only detectable by microscope, while T3bindicates the tumor can be seen or felt by a physician). T4a indicatesthe tumor has grown into the stroma of the prostate in men, and intoeither the uterus or vagina in women. T4b indicates the tumor hasreached the pelvic or abdominal wall. N categories refer to the spreadin the lymph nodes near the pelvis and along the common iliac artery—N0:There is no regional lymph node spread; N1: The cancer has spread to asingle lymph node in the true pelvis; N2: The cancer has spread to 2 ormore lymph nodes in the true pelvis; N3: The cancer has spread to lymphnodes along the common iliac artery. M categories refer to spreadthroughout the body—M0 indicates there are no signs of distant spreadand M1 that cancer has spread to distant parts of the body, e.g.,distant lymph nodes, bones, lungs, liver, etc.

Terconazole may be administered to patients at various stages of bladdercancer. For instance, terconazole may be administered to a patient atStage 0a (Ta, N0, or M0), Stage 0 is (Tis, N0, or M0), Stage I (T1, N0,or M0), Stage II (T2a or T2b, N0, or M0), Stage III (T3a, T3b, or T4a,N0, M0), or Stage IV. In some embodiments, terconazole can beadministered to patients exhibiting symptoms of bladder cancer that havea genetic predisposition to bladder cancer. For instance, the patientmay be SPARC expression positive or negative, or possess one or moremutations in NFL, p53, MIB-1, FEZ1/LZTS1, PTEN, DBCCR1, CDKN2A/MTS1/P6,ERBB2, CDKN2B/INK4B/P15, TSC1, or HRAS1.

Terconazole may be used for the treatment of bladder cancer in mammals,especially humans, in monotherapy mode or in a combination therapy(e.g., dual combination, triple combination etc.) mode such as, forexample, in combination with one or more anti-cancer therapeutics. Insome instances, terconazole, either alone or in combination therapy, canbe administered to a patient that has already undergone a course of BCGtherapy. In some embodiments, the patient may receive BCG treatment 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to commencingterconazole treatment. In other embodiments, terconazole, either aloneor in combination therapy, can be administered to a patient that has notundergone a course of BCG therapy. In yet further embodiments,terconazole, either alone or in combination therapy, can be administeredto a patient that is concurrently undergoing a course of BCG therapy.

Terconazole can be administered to bladder cancer patients alsoreceiving one or more immunotherapeutic agents. Immunotherapies includemonoclonal antibodies, i.e., checkpoint inhibitors, and oncolytic virus.Oncolytic viruses are genetically engineered or naturally occurringviruses that selectively replicate in and kill cancer cells withoutharming the normal tissues. The viruses are modified such that they canreplicate in cancerous cells, but not healthy cells.

The term “anti-cancer drug” is used in broad sense to include, but isnot limited to, oncolytic viruses, monoclonal antibodies, microtubuleinhibitors, topoisomerase inhibitors, platins, alkylating agents, andanti-metabolites. Particular agents include modified adenovirus,modified herpes simplex virus, modified reovirus, modified vacciniavirus, atezolizumab, durvalumab, nivolumab, pembrolizumab, ramucirumab,B-701, MK-6018, ALT-801, paclitaxel, gemcitabine, doxorubicin,vinblastine, etoposide, 5-fluorouracil, carboplatin, oxaliplatin,nedaplatin, altretamine, aminoglutethimide, amsacrine, anastrozole,azacitidine, bleomycin, busulfan, carmustine, chlorambucil,2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide,cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel,estramustine phosphate, floxuridine, fludarabine, gentuzumab,hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon,irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin,procarbazine, rituximab, streptozocin, tamoxifen, temozolomide,teniposide, 6-thioguanine, thiotepa, topotecan, trastuzumab,vincristine, vindesine, and vinorelbine. In certain embodiments, theN-phenyl piperazine can be administered in combination with cisplatin.

In cases of combination therapy, it is possible that a unitary dosageform containing both terconazole and additional anti-cancer agent may beemployed. In some instances, the combinations may be provided in formsuitable for parenteral application such as but not limited toinjection.

In some embodiments, terconazole can be administered as part of asurgical or radiological treatment regime. For instance, a patient maybe administered terconazole prior to and/or after undergoing TURBT,partial or radical cystectomy. Likewise, a patient may be administeredterconazole prior to and/or after undergoing radiation therapy.

Terconazole can be administered as part of a treatment regime thatincludes surgical and chemotherapeutic components. The patient, inaddition to receiving one or more of the anti-cancer agents identifiedabove, can receive terconazole prior to and/or after undergoing asurgical procedure. In some embodiments, terconazole can be administeredas part of a treatment regime that includes radiation therapy andchemotherapeutic components. The patient, in addition to receiving oneor more of the anti-cancer agents identified above, can receiveterconazole prior to and/or after undergoing radiation therapy. In someembodiments, the chemotherapy includes one or more of cisplatin,fluorouracil, and mitomycin. In other embodiments, terconazole can beadministered as part of a treatment regime that includes surgical andimmunotherapeutic components. The patient, in addition to receiving oneor more of the immunotherapeutic agents identified above, can receiveterconazole prior to and/or after undergoing a surgical procedure.

EXAMPLES

The following examples are set forth below to illustrate the methods andresults according to the disclosed subject matter. These examples arenot intended to be inclusive of all aspects of the subject matterdisclosed herein, but rather to illustrate representative methods,compositions, and results. These examples are not intended to excludeequivalents and variations of the present invention, which are apparentto one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures, and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Example 1: Bladder Carcinoma Efficacy Models

Human bladder cancer cell lines HTB-5, HT-1376 and T-24 were purchasedfrom American Type Culture Collection, USA. Cell lines HTB-5 and HT1376were routinely cultured in Eagle's Minimum Essential Medium with 10%fetal bovine serum and T-24 in McCoy's 5A medium with 10% fetal bovineserum. All cell lines were maintained at 37° C. in a humidifiedatmosphere containing 5% CO₂.

In vitro cytotoxicity assay: Cells were plated in 96-well cell cultureplates (4×10³ cells/well) and were treated with drugs at variousconcentrations. Treated cells were maintained at 37° C. in 5% CO₂ for 48and 72 hrs. After the incubation antiproliferative activity of testcompounds was measured using ProMega Cell Titer aqueous one solutioncell proliferation assay kit, viz. CellTiter 96® AQueous One SolutionReagent. Cells were incubated for 1-4 hours at 37° C., 5% CO₂ incubatorand absorbance was measured at 490 nM using a plate reader. IC₅₀ valueswere determined by plotting compound concentration versus cellviability.

Clonogenic assay: bladder cancer cell lines viz HTB-5, HT-1376 and T-24cells were plated at different densities i.e. 300 cells/well and 400cells/well respectively in 3 ml volume per well into six-well plates andincubated overnight at 37° C. in a 5% CO₂ incubator. Next day, cellswere treated with IC₅₀ and 3×IC₅₀ concentrations of ReD for 24 h. Mediumwith drug was then replaced with medium without drug and plates werefurther incubated till visible colonies appeared (approximately for12-14 days). Cells were then fixed with methanol:glacial acetic acid(2:1) and stained with 0.5% crystal violet. After staining for 2-3 min,plates were rinsed twice with D/W, dried and images were captured.

Terconazole showed significant anticancer activity when tested in vitroat varied concentrations against human bladder carcinoma cell lines.T-24, HTB-5 and HT1376.

Example 2: In Vitro 2D Assay

The in-vitro anti-tumor activity of terconazole was assessed in fiveselected human bladder cancer cell lines. The test compounds wereapplied at 9 concentrations in half-log increments. As a referencecompound cisplatin was tested in parallel. Cells were treated for 96 hwith the test compounds. Anti-tumor activity was assessed by using theCellTiter-Blue® Cell Viability Assay. Potency is expressed as absoluteIC₅₀ and relative IC₅₀ values, calculated by non-linear regressionanalysis.

Individual IC₅₀ values for terconazole were in the range from 29.406 μM(BXF 1036) and 46.917 μM (BXF 1218 μM).

The reference compound cisplatin showed concentration-dependent activityin all cell lines tested with a geometric mean absolute IC₅₀ value of8.573 μM.

The in-vitro anticancer activity of terconazole was assessed. Antitumoractivity was assessed in five selected bladder cancer cell lines byusing the CellTiter-Blue® Cell Viability Assay.

Tumor Cell Lines: In the present study the human bladder cancer celllines BXF 1036, BXF 1218, BXF 1352, 5637 and T24 were used. BXF 1036,BXF 1218 and BXF 1352 were established at Oncotest from thecorresponding human patient derived xenograft. T24 was purchased fromATCC (Rockville, Md., USA) and 5637 was from DSMZ (Braunschweig,Germany). Authenticity of cell lines was confirmed at the DSMZ by STR(short tandem repeat) analysis, a PCR based DNA-fingerprintingmethodology. Cell lines were routinely passaged once or twice weekly andmaintained in culture for up to 20 passages. All cells were grown at 37°C. in a humidified atmosphere with 5% CO₂ in RPMI 1640 medium (25 mMHEPES, with L-glutamine, #FG1385, Biochrom, Berlin, Germany)supplemented with 10% (v/v) fetal calf serum (Sigma, Taufkirchen,Germany) and 0.1 mg/mL gentamicin (Life Technologies, Karlsruhe,Germany).

Cell Proliferation Assay: The CellTiter-Blue® Cell Viability Assay(#G8081, Promega) was used according to manufacturer's instructions.Briefly, cells were harvested from exponential phase cultures,cells/well depending on the cell line's growth rate. After a 24 hrecovery period to allow the cells to resume exponential growth, testcompounds were added. Compounds were applied at 9 concentrations inhalf-log increments in duplicate and treatment continued for 96 h. After96 h treatment of cells, 20 μL/well CellTiter-Blue® reagent was added.

Following an incubation period of up to four hours, fluorescence (FU)was measured by using the Enspire Multimode Plate Reader (excitationk=531 nm, emission k=615 nm). For calculations, the mean values ofduplicate/sixfold (untreated control) data were used. Sigmoidalconcentration-response curves were fitted to the data points (T/Cvalues) obtained for each cell line using 4 parameter non-linear curvefit (Oncotest Warehouse Software).

The in-vitro anti-tumor activity of terconazole was assessed in fiveselected human bladder cancer cell lines by using CellTiter-Blue®

Cell Viability Assay: Concentration-dependent activity was also detectedfor terconazole in all cell lines tested with a geometric mean absoluteIC₅₀ value of 39.872 μM. Individual IC₅₀ values for terconazole were inthe range from 29.406 μM (BXF 1036) and 46.917 μM (BXF 1218), indicatinga low level of selective activity.

The reference compound cisplatin showed concentration-dependent activityin all cell lines tested with a geometric mean absolute IC₅₀ value of8.573 μM. The selectivity profile of cisplatin was quite similar toCIPDE034 (terconazole), with T24 shown to be the most sensitive and BXF1352 the most resistant cell line. The IC₅₀ values are tabulated in thebelow table.

Absolute IC₅₀ values (μM) Cell Line Terconazole Cisplatin BXF 103629.406 9.529 BXF 1218 46.917 7.078 BXF 1352 45.864 17.059 BXF 563738.368 9.706 BXF T24 41.508 4.148 Geometric Mean Absolute IC₅₀ values(μM) 39.872 8.573

Example 3: In Vitro 3D Assay

In the present study, terconazole and cisplatin were evaluated for theirability to inhibit anchorage independent growth and ex vivo colonyformation of tumor cells in semi-solid medium. The compounds were testedagainst 5 human tumor cell lines of bladder cancer. Test concentrationsranged, from 0.0316 μM to 316.2 μM (terconazole), or from 0.00316 μM to100 μM (cisplatin).

Terconazole and cisplatin inhibited colony formation in aconcentration-dependent manner. Terconazole inhibited colony formationof all bladder cancer cell lines tested with IC₅₀ values ranging from29.56 μM to 40.05 μM. IC₅₀ values were very similar, thus differentialanti-tumor efficacy was not that pronounced. Bottom plateaus of theconcentration-effect curves of responding tumor models were 0%,indicating clear inhibition of tumor colony growth.

Cisplatin inhibited colony formation with a mean relative IC₅₀ value of9.71 μM (mean absolute IC₅₀ value=9.93 μM). Bottom plateaus of theconcentration-effect curves of the responding tumor models were <10%,indicating clear inhibition of tumor colony growth. Based on relativeIC₅₀ values, above average activity was observed against ⅛ tumor models(cell line BXF 1036).

Terconazole and the reference compound cisplatin were investigated foranticancer activity ex vivo in five human tumor cell lines of bladdercancer. Tests were carried out using a 3D clonogenic assay in 96-wellformat with image based read-out. The aim of the study was toinvestigate antitumor potency and tumor type selectivity of thecompound.

Clonogenic Assay Procedure: The clonogenic assay was carried out in a 96well plate format using ultra low attachment plates. For each test,cells were prepared as described above and assay plates were prepared asfollows: each test well contained a layer of semi-solid medium withtumor cells (50 and a second layer of medium supernatant with or withouttest compound (100 The cell layer consisted of 2·10³ to 3·10³ tumorcells per well, which were seeded in 50 μL/well cell culture medium(IMDM, supplemented with 20% (v/v) fetal calf serum, 0.01% (w/v)gentamicin, and 0.4% (w/v) agar. After 24 hours the test compounds wereadded after serial dilution in cell culture medium, and left on thecells for the duration of the experiment (continuous exposure, 100 μldrug overlay). Every plate included six untreated control wells anddrug-treated groups in duplicate at 9 concentrations. Cultures wereincubated at 37° C. and 7.5% CO₂ in a humidified atmosphere for 8 to 13days and monitored closely for colony growth using an invertedmicroscope. Within this period, ex vivo tumor growth led to theformation of colonies with a diameter of >50 μm. At the time of maximumcolony formation, counts were performed with an automatic image analysissystem (Bioreader 5000-Wa Biosys GmbH). 48 hours prior to evaluation,vital colonies were stained with a sterile aqueous solution of2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1mg/ml, 100 μl/well)

Terconazole inhibited colony formation in a concentration-dependentmanner. The mean relative IC₅₀ value was determined as 33.48 μM (meanabsolute IC₅₀ value=33.88 μM). Bottom plateaus of theconcentration-effect curves of responding tumor models were 0%,indicating clear inhibition of tumor colony growth. IC₅₀ values werevery similar, thus differential anti-tumor efficacy was not thatpronounced.

The IC₅₀ values are tabulated in the table below.

Absolute IC₅₀ values (μM) Cell Line Terconazole Cisplatin BXF 1036 29.673.97 BXF 1218 40.27 5.36 BXF 1352 36.59 8.65 BXF 5637 30.80 10.08 BXFT24 33.15 52.99 Geometric Mean Absolute IC₅₀ values (μM) 33.88 9.93

Example 4: In Vitro Combination Study

Summary: Terconazole was tested alone and in combination with cisplatinin order to investigate the ability to inhibit tumor cell growth ofbladder cancer cell lines in a 5×5 matrix combination format. Efficacyof the combinations was assessed by measuring anchorage-independentgrowth and in vitro tumor colony formation using a 3D clonogenic assayin cell lines BXF 1036L, BXF 1218L, and BXF T24.

Terconazole tested as single agent inhibited colony formation of tumorcells seeded in soft-agar in a concentration-dependent manner with IC₅₀values ranging from 27.1 μM (BXF 1036L) to 59.8 μM (BXF T24). Cisplatinwas active against the cell lines BXF 1036L and BXF 1218L (IC₅₀ of 6.09μM and 6.8 respectively) while being less active against BXF T24(IC₅₀=77.4 μM).

Additive effects were observed for the combination of terconazole withcisplatin in bladder cancer cell lines BXF 1036L, BXF 1218L, and BXFT24.

The objective of this study was to assess anti-tumor efficacy ofCIPDE033 and

CIPDE034 in combination with cisplatin in a 5×5 matrix combinationformat against various bladder cancer cell lines. Efficacy of thecombinations was assessed by measuring anchorage-independent growth andin vitro tumor colony formation using a 3D clonogenic assay in celllines BXF 1036L, BXF 1218L, and BXF T24. The Bliss independencemethodology was used for data analysis, in order to identify possiblesynergistic effects.

The compounds were tested in ovarian cell lines, namely BXF 1036L, BXF1218L, and BXF T24. Cells lines of BXF 1036L and BXF 1218L wereestablished at Oncotest in Freiburg from the correspondingpatient-derived xenografts. BXF T24 cells were obtained from AmericanType Culture Collection (Rockville, Md., USA). Authenticity of celllines was confirmed at the DSMZ by STR analysis.

Cultivation of Cell Lines: Cell lines were routinely passaged once ortwice weekly and maintained in culture for up to 20 passages. Cells weregrown at 37° C. in a humidified atmosphere with 5% CO₂ in RPMI 1640medium (25 mM HEPES, with L-glutamine, Biochrom) supplemented with 10%(v/v) fetal calf serum and 0.1 mg/mL gentamicin. The percentage ofviable cells was determined in a Neubauer-hemocytometer using trypanblue exclusion.

3D Clonogenic Assay Procedure: The clonogenic assay was carried out in a96 well plate format using ultra low attachment plates. For each testcells were prepared as described above, and assay plates were preparedas follows: each test well contained a layer of semi-solid medium withtumor cells (50 μl), and a second layer of medium supernatant with orwithout test compounds (100 μl). The cell layer consisted of 5·10³ to7.5·10³ tumor cells per well, which were seeded in 50 μl/well cellculture medium (IMDM, supplemented with 20% (v/v) fetal calf serum,0.01% (w/v) gentamicin, and 0.4% (w/v) agar). After 24 h, the soft-agarlayer was covered with 90 μl of the same culture medium without agar,and compounds were added after serial dilution in DMSO and transfer incell culture medium and left on the cells for the duration of theexperiment (continuous exposure, 100 μL total drug overlay). Every plateincluded six untreated control wells and drug-treated groups. Cultureswere incubated at 37° C. and 7.5% CO₂ in a humidified atmosphere for 8to 13 days and monitored closely for colony growth using an invertedmicroscope. Within this period, ex vivo tumor growth led to theformation of colonies with a diameter of >50 μm. At the time of maximumcolony formation, counts were performed with an automatic image analysissystem (CellInsight NXT, Thermo Scientific). 48 hours prior toevaluation, vital colonies were stained with a sterile aqueous solutionof 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1mg/ml, 100 μl/well).

Results and Discussion: The ability of terconazole to inhibit ex vivocolony formation of cells as single agent and in combination withcisplatin was examined in 3 bladder cancer cell lines (BXF 1036L, BXF1218L and BXF T24). Information about single agent efficacy was derivedfrom monotherapy controls of the 5×5 combination matrix. Terconazoletested as single agent inhibited colony formation of tumor cells seededin soft-agar in a concentration-dependent manner with IC₅₀ valuesranging from 27.1 μM (BXF 1036L) to 59.8 μM (BXF T24). Cisplatin wasactive against the cell lines BXF 1036L and BXF 1218L (IC₅₀ of 6.09 μMand 6.8 μM, respectively) while being less active against BXF T24(IC₅₀=77.4 μM). Additive effects were observed for the combination ofterconazole with cisplatin in bladder cancer cell lines BXF 1036L, BXF1218L, and BXF T24.

Terconazole and cisplatin inhibited colony formation of BXF 1036L cellsseeded in soft agar in a concentration-dependent manner. This is alsoreflected in the matrix combination, where activity of the differentcombinations was observed at higher concentrations of both compounds.

Bliss independence analysis showed that overall an additive effect ofthe combinations was obtained, i.e. neither synergy nor antagonism. Thecolor coding of the tiles in the heatmap show that there is noconsistent concentration-dependent effect pointing towards synergy(BI>0.15) or antagonism (BI<−0.15). The results are depicted in FIG. 5.

Terconazole and cisplatin inhibited colony formation of BXF 1218L cellsseeded in soft agar in a concentration-dependent manner. This is alsoreflected in the matrix combination, where activity of the differentcombinations was observed at higher concentrations of both compounds

Bliss independence analysis showed that overall an additive effect ofthe combinations was obtained, i.e. neither synergy nor antagonism. Thecolor coding of the tiles in the heatmap show, that there is noconsistent concentration-dependent effect pointing towards synergy(BI>0.15) or antagonism (BI<−0.15). The results are depicted in FIG. 6.

Terconazole and cisplatin inhibited colony formation of BXF T-24 cellsseeded in soft agar in a concentration-dependent manner. This is alsoreflected in the matrix combination, where activity of the differentcombinations was observed at higher concentrations of both compounds.

Bliss independence analysis showed that overall an additive effect ofthe combinations was obtained, i.e. neither synergy nor antagonism. Thecolor coding of the tiles in the heatmap show, that there is noconsistent concentration-dependent effect pointing towards synergy(BI>0.15) or antagonism (BI<−0.15). The results are depicted in FIG. 7.

Example 5: Compositions

Terconazole Soft Gelatin Capsule Ingredient Qty. mg/unit Terconazole 20-400 D-alpha-tocopheryl polyethylene 250-550 glycol 1000 succinate(TPGS) Polyethylene glycol 400 250-400 Polyethylene glycol 400 25-40

-   1. Load D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS)    in a suitable stainless steel jacketed vessel and heat to 50° C.    until liquefied.-   2. Heat Polyethylene glycol 400 (90%) in a separate stainless steel    vessel to 50° C. and add to the liquefied material of step 1 slowly.-   3. Mix the ingredients of step 2 using a suitable stirrer until    homogenous solution obtained.-   4. Increase the temperature of the homogenous solution to 65° C.,    and add Terconazole to the solution of step 3 under constant    stirring and dissolved.-   5. Add remaining quantity of Polyethylene glycol 400 (10%) to the    solution of step 4 and cool the solution to room temperature.-   6. Apply vacuum to remove the entrapped air.-   7. Fill mixture using in white opaque soft gelatin capsules (size    12, oblong) using a capsule-filling machine. The capsules were    further dried to a moisture level of 3-6% and shell hardness of 7-10    N and packed in a suitable container.    -   Terconazole tablets (Hot Melt Extrusion technique)

TABLE 2A (Terconazole Granule) Ingredients Formula I (%) Formula II (%)Formula III (%) Terconazole 100 100 100 Poloxamer 407 70 80 90 SentryPolyox WSR 30 20 10 N80 (PEO)

TABLE 2 B (Terconazole Tablet) Ingredients Qty/Tab (mg) Terconazolegranules (HME)  20-300 Lactose monohydrate.  40-120 MacrocrystallineCellulose  75-225 Hypromellose 60-90 Croscarmellose sodium 15-45Colloidal anhydrous silica 2-6 Magnesium stearate 1-5 Talc 1-5 Opadryready mix 10-20 Purified water Qs

Procedure: The composition as covered under Table 2 is prepared usingfollowing process:

Terconazole Granule:

-   1. Use hot melt extruder (Make: Thermo Scientific Instruments) in    the preparation of extrudates-   2. Shift all powders through specified mesh-   3. Blend the powders of step 1 in V-shell blender prior to extrusion-   4. Blend the powder of step 2 in the hopper located at the head of    vertical screw such that the material is flood fed by gravity for    residence time of approximately 3 minutes.-   5. Shear the extrudate of step 4 after exiting the die and allowed    to cool at ambient conditions.-   6. Pass the extrudates of step 5 again through quadro co-mill to get    granules of desired size.

Terconazole Tablet:

-   1. Blend Terconazole granules obtained in part A further in V-shell    blender with lactose monohydrate, microcrystalline cellulose,    hypromellose, croscarmellose sodium, colloidal anhydrous silica, and    talc previously passed through suitable sieves.-   2. Lubricate the blend in a V-shell blender with magnesium stearate    previously sifted through suitable sieve.-   3. Compress the lubricated blend of step 2 into tablets using    suitable tooling using a tablet compression machine.-   4. Coat the tablets using a film coating dispersion. (The film    coating dispersion was prepared by dispersing the opadry ready mix    in purified water).

Terconazole tablets Ingredients Quantity mg/tablet Terconazole 20-300Lactose monohydrate 30-150 Microcrystalline cellulose (Avicel PH 101)40-160 Pregelatinized starch 30-60  Croscarmellose sodium 15-45 Poloxamer 188 (Pulmonic F 68) 5-20 Silicon dioxide colloidal 2.5-10 Magnesium stearate 3-10 Purified water q. s

-   1. Shift Terconazole, lactose monohydate, pregelatinized starch, and    a portion (one-half) of croscarmellose sodium in a mixer through #30    sieve.-   2. Load the sifted powders of step 1 in a suitable mixer/granulator    and mix the materials for 20 minutes.-   3. Dissolve poloxamer 188 in sufficient quantity of purified water,    and use it to wet granulate the blend of step 2.-   4. Dry the granules of step 3 in a fluidized-bed dryer until the LOD    is 2% or less.-   5. Pass the dried granules of step 4 through a screen, or mill them    to obtain granules of the desired size (1-3 mm).-   6. Blend the sized granules of step 5 with silicon dioxide    (previously sifted through #60 Sieve), microcrystalline cellulose    (pre sifted through #30 sieve), and the remaining croscarmellose    sodium in octagonal blender for 7 minutes.-   7. Lubricate the blend of step 6 by adding magnesium stearate    (previously sifted through #60 Sieve) to the blend of step 6 and    further blending for 3 minute.-   8. Compress lubricated blend of step 7 into tablets using suitable    tooling using a tablet compression machine.

The compositions and methods of the appended claims are not limited inscope by the specific compositions and methods described herein, whichare intended as illustrations of a few aspects of the claims and anycompositions and methods that are functionally equivalent are intendedto fall within the scope of the claims. Various modifications of thecompositions and methods in addition to those shown and described hereinare intended to fall within the scope of the appended claims. Further,while only certain representative compositions and method stepsdisclosed herein are specifically described, other combinations of thecompositions and method steps also are intended to fall within the scopeof the appended claims, even if not specifically recited. Thus, acombination of steps, elements, components, or constituents may beexplicitly mentioned herein or less, however, other combinations ofsteps, elements, components, and constituents are included, even thoughnot explicitly stated. The term “comprising” and variations thereof asused herein is used synonymously with the term “including” andvariations thereof and are open, non-limiting terms. Although the terms“comprising” and “including” have been used herein to describe variousembodiments, the terms “consisting essentially of” and “consisting of”can be used in place of “comprising” and “including” to provide for morespecific embodiments of the invention and are also disclosed. Other thanin the examples, or where otherwise noted, all numbers expressingquantities of ingredients, reaction conditions, and so forth used in thespecification and claims are to be understood at the very least, and notas an attempt to limit the application of the doctrine of equivalents tothe scope of the claims, to be construed in light of the number ofsignificant digits and ordinary rounding approaches.

What is claimed is:
 1. A method for the treatment of bladder cancer in apatient, the method comprising administering to said patientterconazole, or a pharmaceutically acceptable salt thereof, in an amounteffective to treat bladder cancer, wherein the patient is diagnosed withStage 0, Stage I, Stage II, Stage III or Stage IV bladder cancer,wherein the terconazole is administered as a single agent or incombination with an anti-cancer agent selected from the group consistingof oncolytic viruses, monoclonal antibodies, microtubule inhibitors,topoisomerase inhibitors, platins, alkylating agents, andantimetabolites.
 2. The method according to claim 1, wherein the patientalso receives surgical or radiation therapy.
 3. The method according toclaim 1, wherein the patient also receives Bacillus Calmette-Guérinvaccine.
 4. The method according to claim 3, wherein terconazole isadministered subsequent to Bacillus Calmette-Guérin vaccine delivery. 5.The method according to claim 1, wherein terconazole is administered incombination with an immunotherapy comprising a monoclonal antibody or aoncolytic virus.
 6. The method according to claim 3, wherein the surgerycomprises transurethral resection of bladder tumor (TURBT), partial orradical cystectomy.
 7. The method according to claim 6, whereinterconazole is administered subsequent to surgical treatment for bladdercancer.
 8. The method according to claim 6, wherein terconazole isadministered prior to the surgical treatment for bladder cancer.
 9. Themethod according to claim 1, wherein the anti-cancer agent comprises aplatin compound.
 10. The method according to claim 9, wherein platincompound comprises cisplatin, carboplatin, oxaliplatin, or nedaplatin.11. The method according to claim 10, wherein platin compound iscisplatin.
 12. A kit comprising terconazole and at least one anti-canceragent comprising one or more monoclonal antibodies, oncolytic viruses,microtubule inhibitors, topoisomerase inhibitors, platins, alkylatingagents, or anti-metabolites.